xcex949-Tetrahydrocannabinol, the pyschoactive marijuana derived cannabinoid, binds to the CB1 receptor in the brain and to the CB2 receptor in the spleen. Compounds which stimulate the CB1 receptor have been shown to induce analgesia and sedation, to cause mood elevation, to control nausea and appetite and to lower intraocular pressure (Mechoulam, Cannabinoids as Therapeutic Agents, CRC Press, Boca Raton, Fla. (1986), Fride and Mechoulam, Eur. J. Pharmacol. 231:313 (1993), Crawley et al., Pharmacol. Biochem. Behav. 46:967 (1993) and Smith et al., J. Pharm. Exp. Therap. 270:219 (1994)). Cannabinoids have also been shown to suppress the immune system (Mechoulam, Cannabinoids as Therapeutic Agents, CRC Press, Boca Raton, Fla. (1986). Thus, compounds which stimulate the CB1 or CB2 receptor, directly or indirectly, are potentially useful in treating glaucoma, preventing tissue rejection in organ transplant patients, controlling nausea in patients undergoing chemotherapy, controlling pain and enhancing the appetite and controlling pain in individuals with AIDS Wasting Syndrome.
Arachidonyl ethanolamide (anandamide) is a naturally-occurring brain constituent that acts as a CB1 and CB2 agonist and exhibits pharmacological activity in mice comparable to cannabinoids (Fride and Mechoulam (1993), Crawley et al. (1993) and Smith et al. (1994)). Anandamide is cleaved in vivo by anandamide amidase. Thus, inhibitors of anandamide amidase have the effect of indirectly stimulating the CB1 and CB2 receptors by increasing in vivo levels of anandamide.
In addition to acting at the CB1 and CB2 receptors, cannabinoids also affect cellular membranes, thereby producing undesirable side effects such as drowsiness, impairment of monoamide oxidase function and impairment of non-receptor mediated brain function. The addictive and psychotropic properties of cannabinoids also limit their therapeutic value. Inhibitors of anandamide amidase are not expected to have the undesired membrane-related side-effects produced by cannabinoids. By providing an alternate mechanism for stimulating the CB1 and CB2 receptor, anandamide inhibitors might not have the addictive and psychotropic properties of cannabinoids. However, present inhibitors of anandamide amidase have disadvantages. For example, phenylmethylsulfonyl fluoride (PMSF) is toxic to cells. Thus, there is a need for new and more potent inhibitors of anandamide amidase which have reduced toxicity towards cells and which do not significantly interact with the CB1 or CB2 receptor at inhibitory concentrations.
It has now been found that long chain fatty acids and aromatic acid analogs of long chain fatty acids with head groups capable of irreversibly binding to a nucleophilic group at an enzyme active site are potent inhibitors of anandamide amidase. For example, palmitylsulfonyl fluoride was found to increase the level of undegraded anandamide 55-fold at 10 nM in intact neuroblastoma cells (Example 1) and is therefore more than 100 fold more potent than phenylmethylsulfonyl fluoride at inhibiting anandamide amidase. At the same time, the inhibitors disclosed herein have a low affinity for the CB1 receptor (Example 3). For example, the binding affinity of palmitylsulfonyl fluoride for the CB1 receptor is about 10 times lower than anandamide. In addition, it has been found that palmitylsulfonyl fluoride causes some of the same pharmacological effects in rats as do compounds which stimulate the CB1 receptor directly, such as xcex949-tetrahydrocannabinol. For example, palmitylsulfonyl fluoride is shown herein to induce analgesia in rats (Example 4). Based on these results, methods of inhibiting anandamide amidase, thereby stimulating the CB1 and CB2 receptors, in an individual or animal are disclosed. Also disclosed are novel compounds which inhibit anandamide amidase.
The present invention is a method of inhibiting anandamide amidase in an individual or animal. The method comprises administering to the individual or animal a therapeutically effective amount of a compound represented by Structural Formula I:
Rxe2x80x94Xxe2x80x94Y xe2x80x83xe2x80x83(I) 
and physiologically acceptable salts thereof.
R is selected from the group consisting of a methyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a heterocyclic group and a substituted heterocyclic group.
X is a straight chain hydrocarbyl group or a substituted straight chain hydrocarbyl group containing from about 4 to about 18 carbon atoms if R is an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a heterocyclic group or a substituted heterocyclic group.
X is a hydrocarbyl group or a substituted hydrocarbyl group containing from about 10 to about 24 carbon atoms if R is a methyl group.
Y is a moiety capable of irreversibly binding with a nucleophilic group at the active site of an amidase enzyme.
The method and the novel compounds disclosed herein have therapeutic uses. For example, the compounds and methods of the present invention, like cannabinoids, can relieve the pain caused by cancer and the nausea resulting from cancer chemotherapy. They are not expected to have the undesirable membrane-related side-effects associated with cannabinoids. In addition, the methods and compounds disclosed herein are expected to be immunosuppressive and can therefore be used to prevent organ rejection in an individual undergoing an organ transplant. Because the compounds and methods of the present invention enhance the appetite of an individual, they can be used treat patients with AIDS Wasting Syndrome, who are often suffering from malnourishment as a result of appetite loss.
The novel inhibitors of anandamide amidase disclosed herein also have research uses. For example, they can be used to maintain the level of anandamide in vitro to study the effect of ananamide on cells and to maintain the level of anandamide in vivo to study the effect of anandamide on individuals and animals. They can be used to characterize cells, for example to determine if a cell type has cannabimetic or amidase activity. For example, the inhibitors can be used to determine if a cell population expresses anandamide amidase by contacting the cells with an inhibitor and then determining if there is an increase in the concentration of anandamide. The anandamide inhibitors disclosed herein can also be used as in aid in drug design, for example as a control in assays for testing other compounds for their ability to inhibit anandamide amidase and to determine the structure activity requirements of anandamide amidase inhibitors.